NOX enzymes: potential target for the treatment of acute lung injury

MULTI-AUTHOR REVIEW

NOX enzymes: potential target for the treatment of acute lunginjury

Stephanie Carnesecchi • Jean-Claude Pache •

Constance Barazzone-Argiroffo

Received: 18 April 2012 / Revised: 18 April 2012 / Accepted: 20 April 2012 / Published online: 13 May 2012

� Springer Basel AG 2012

Abstract Acute lung injury (ALI) and its more severe

form, acute respiratory distress syndrome (ARDS), is char-

acterized by acute inflammation, disruption of the alveolar-

capillary barrier, and in the organizing stage by alveolar

pneumocytes hyperplasia and extensive lung fibrosis. The

cellular and molecular mechanisms leading to the develop-

ment of ALI/ARDS are not completely understood, but there

is evidence that reactive oxygen species (ROS) generated by

inflammatory cells as well as epithelial and endothelial cells

are responsible for inflammatory response, lung damage, and

abnormal repair. Among all ROS-producing enzymes, the

members of NADPH oxidases (NOXs), which are widely

expressed in different lung cell types, have been shown to

participate in cellular processes involved in the maintenance

of lung integrity. It is not surprising that change in NOXs’

expression and function is involved in the development of

ALI/ARDS. In this context, the use of NOX inhibitors could

be a possible therapeutic perspective in the management of

this syndrome. In this article, we summarize the current

knowledge concerning some cellular aspects of NOXs

localization and function in the lungs, consider their contri-

bution in the development of ALI/ARDS and discuss the

place of NOX inhibitors as potential therapeutical target.

Keywords NOX enzymes � Acute lung injury �Acute respiratory distress syndrome

Abbreviations

ALI Acute lung injury

ARDS Acute respiratory distress syndrome

BAL Bronchoalveolar lavage

ROS Reactive oxygen species

HM Hyaline membranes

LPS Lipopolysaccharide

TNF-a Tumor necrosis factor-aICAM-1 Intracellular adhesion molecule-1

EC Endothelial cells

TGF-b1 Transforming growth factor-b1

IPF Idiopathic pulmonary fibrosis

IRF-3 Interferon regulatory factor-3

AP1 Activator protein 1

NF-jB Nucleor factor jB

MCP-1 Monocyte chemotactic protein-1

MV Mechanical ventilation

N.D. Not determined

Introduction

Acute lung injury (ALI) and acute respiratory distress

syndrome (ARDS), which is the most severe form, is

associated with a high mortality (50–80 %). ALI/ARDS

affects a large number of patients entering intensive care

units and is defined by bilateral pulmonary infiltrates on

chest radiograph, hypoxemic respiratory failure measured

by a partial pressure of arterial oxygen (PaO2)/fraction of

inspired oxygen (FiO2) ratio (PaO2/FiO2 \ 300 mmHg for

ALI and \200 mmHg for ARDS) with normal hydrostatic

pressure corresponding to the absence of left heart failure.

Acute respiratory distress syndrome can occur with several

diseases either associated with those causing direct lung

injury such as pneumonia, gastric aspiration or toxic

S. Carnesecchi (&) � C. Barazzone-Argiroffo

Department of Pediatrics/Pathology and Immunology,

Centre Medical Universitaire, 1 rue Michel Servet,

1211 Geneva, Switzerland

e-mail: [email protected]

J.-C. Pache

Department of Pathology and Immunology, Centre Medical

Universitaire, 1 rue Michel Servet, 1211 Geneva, Switzerland

Cell. Mol. Life Sci. (2012) 69:2373–2385

DOI 10.1007/s00018-012-1013-6 Cellular and Molecular Life Sciences

123

inhalation, or indirect injury such as sepsis or severe burn.

The heterogeneity of causes and the complexity of clinical

histopathological and radiographic manifestations make

the study of ARDS pathogenesis and the test of new

therapeutics difficult. Indeed, ARDS follows most often a

progressive course characterized by two distinct stages.

The acute (or exudative) stage includes the disruption of

the alveolar-capillary barrier, pulmonary edema, accumu-

lation of protein-rich fluid into the interstitium, alveolar

space, and diffuse inflammation. The later organizing stage

occurs with the resolution of pulmonary edema and lung

inflammation. During this phase, alveolar pneumocytes

hyperplasia and fibroblast proliferation lead to disordered

collagen deposition and extensive lung fibrosis.

Although the elucidation of the cellular and molecular

mechanisms involved in the pathogenesis of ALI/ARDS

remain unclear and complex, there is evidence that reactive

oxygen species (ROS) contribute to the initiation of

endothelial damage characteristic of ARDS and are

responsible for most of clinical symptoms of this syndrome

[1]. Indeed, a large amount of ROS, which are found in

broncho alveolar lavages (BAL) of ARDS patients are

produced mainly by alveolar macrophages, neutrophils,

lung endothelial, and epithelial cells. These ROS can alter

gene and protein function. Among several ROS-producing

enzymes, NADPH oxidase (NOX) enzymes, which are

membrane-bound complexes catalyzing the reduction of

molecular oxygen (O2) to superoxide (O2-) [2], are

involved in principal clinical manifestations of ALI/ARDS

[1, 3]. The first NOX has been described in phagocytes and

is a complex that includes a catalytic subunit gp91phox

called NOX2 associated with p22phox and cytosolic regu-

latory subunits such p47phox, p67phox and small GTPase

(RAC1 or RAC 2), required for NOX activation and gen-

eration of superoxide [2, 4]. Recently, structural

homologues of the phagocyte NOX enzyme were identi-

fied, such as NOX1-3-4-5, DUOX1, and DUOX2. Despite

their similar structure and enzymatic function, NOX

enzymes differ in their mechanisms of activation, which

depend on the recruitment of membrane or/and cytosolic

regulatory subunits such as p22phox, p47phox, p67phox,

NOXO1, NOXA1, and RAC [2]. The NOX isoforms,

which are expressed in a variety of lung cell types [5],

participate in several cellular processes [6] and are

involved in lung pathological situations such as ALI/

ARDS, cancer, fibrosis, pulmonary hypertension, and

obstructive lung disorders such as emphysema, asthma, and

cystic fibrosis [5, 7–10].

In the present article, we will focus on the contribution of

NOX enzymes in the development of ALI/ARDS. We will

first briefly describe some cellular aspects of NOX locali-

zation and function in the lungs. In the second part, we will

review the current knowledge concerning the role of NOX-

dependent ROS production in the pathogenesis of ALI/

ARDS and particularly its involvement in some clinical

aspects of this disease. In the third part, we will discuss their

therapeutic potential in the management of ARDS/ALI.

Cellular expression and function of NOX enzymes

in lungs

The lung, of which the principal function is to deliver

oxygen to tissues, is widely exposed to deleterious envi-

ronmental factors including virus, bacteria, irritants and

allergens, and possesses a potent innate defense system.

This defense system not only uses the phagocyte NOX

system to eliminate these dangers through oxidative killing

[6, 11] but also in regulating cell-signaling pathways

involved in host defense mechanisms, cell proliferation,

migration, and/or differentiation [4, 12]. Several studies

have shown that NOX enzymes are expressed in lungs,

both in mice and in humans. The amount of the different

NOX isoforms depends on the cell types and also on the

species. A high amount of NOX2 [13, 14] and DUOX1/2

mRNA [15, 16] as well as NOX1 [17, 18] and NOX4 [7, 8,

13, 18] are detected in lungs. In addition to the expression

of NOX2 in alveolar macrophages and other inflammatory

cell types, NOX isoforms have been detected in different

lung cell types such as alveolar epithelial and endothelial

cells, fibroblasts, smooth muscle cells, and airway epithe-

lial cells [19, 20]. The cell-type-dependent NOX

expression in lungs suggests their specific participation in

some aspect of physiological and pathological functions

including host defenses, proliferation, migration, and/or

differentiation. The specific lung expression and function

of NOX enzymes are summarized in the Table 1.

Thus, according to the physiological contribution of

NOX enzymes in tissue repair and/or remodeling, we could

envisage that the modulation of their expression and acti-

vation in different lung cell types contributes to the

development of lung diseases such as ALI/ARDS. We will

first describe the pathogenesis of ALI/ARDS and then the

role of ROS-dependent NOX enzymes in different animal

models of ARDS/ALI.

Histopathology and pathogenesis of ALI/ARDS

In spite of the scarce knowledge concerning the mecha-

nisms involved in the pathogenesis of ALI/ARDS,

histological analysis of lung sections from ARDS at dif-

ferent stages suggests that lung modifications occurring

during this disease follow a scheduled time course and can

be divided into three time-dependent phases: acute (or

exudative), proliferative, and fibrosis [61].

2374 S. Carnesecchi et al.

123

The acute phase begins after the initial injury; most cells

composing the alveolar septa undergo either apoptosis or

necrosis, and inflammatory cells mainly represented by the

neutrophils, invade alveolar walls and lumens. The number

of neutrophils increases fast as they travel via the inter-

stitium into the airspaces. This is facilitated by the

disruption of the vascular structures, which occurs when

neutrophils and fibrin plugs occlude the capillaries

(Fig. 1a, b). Hyaline membranes (HM) made of fibrin,

proteins and cellular debris; accumulate along the alveolar

walls (Fig. 1a, b). By electron microscopy, all stages of

epithelial degeneration can be observed from slight cyto-

plasmic swelling to huge blister formation and total

destruction of the epithelial lining [62]. In parallel, the

endothelial cell layer is often irregular because of cyto-

plasmic swelling and large vacuoles. Endothelial defects

Table 1 Summary of NOX enzymes localization, activation, and function in lung cells

NOX

isoforms

Expression Stimuli Function Species References

NOX1 Endothelial cells FGF-b, VEGF Vascular cell growth M, H [21]

FGF-b, VEGF Angiogenesis M, H [21]

Hyperoxia Cell death M [17]

Alveolar epithelial

cells

TNF-a, hyperoxia Cell death M [17, 22]

Hypoxia HIF-a signaling H [23, 24]

Growth factors, HIPK2 depletion Proliferation M, H [25, 26]

Fibroblasts N.D N.D M Personal

data

Vascular smooth

muscle cells

– N.D R [27]

NOX2 Endothelial cells Hyperoxia Cell migration H [28, 29]

Ischemia and High K?, hypoxia Oxygen sensing B, M [30–33]

LPS, TNF-a TLR2 crosstalk M [34]

Neuro-epithelial

cells

Hypoxia Chemoreceptor O2 sensing H, R,

Ra

[35]

Macrophages/

neutrophils

TNF-a, LPS influenza A virus Anti-microbial host defense/innate

immune response

M, H [34, 36–41]

Chronic fine particulate TLR4 crosstalk, NF-jB activation M [42]

NOX3 Endothelial cells – TLR4 crosstalk M [10]

Hyperoxia Cell integrity M [43]

NOX4 Endothelial cells Hyperoxia Cell migration H [28, 44]

Alveolar epithelial

cells

Bleomycin, TGF-b1, fine particles Cell death M, H [7, 45]

Smooth muscle

cells

TGF-b1 Proliferation M, H [18, 46, 47]

Differentiation R [27]

Fibroblasts/

myofibroblasts

Bleomycin, TGF-b1, radiation Differentiation/activation M, H [7, 8, 48, 49]

Hypoxia Proliferation H [50]

DUOX1 Bronchial cells Pseudomonas aeruginosa, LPO, IL-4, IL-13

cytokines, and cigarette smoke

Host defense H [15, 19, 20,

51–54]

PMA, human neutrophil elastase Mucin expression H [55]

ATP Cellular migration H [56]

– H? production and secretion H [57]

LPS Cell proliferation M [58]

Alveolar epithelial

cells

Hormone mixture Differentiation H [59]

DUOX2 Bronchial cells IFN-c Host defense H [19, 60]

– H? production and secretion H [57]

Alveolar epithelial

cells

– N.D M Personal

data

M mouse, H human, R rat, Ra rabbit, B bovine, PMA phorbol 12-myristate 13-acetate, HIPK2 homeo domain-interacting protein kinase-2, LPOlactoperoxidase, ATP adenosine triphosphate, ANPH atrial natriuretic peptide hormone, N.D not determined

NOX enzymes and acute lung injury 2375

123

are covered by fibrin or microthrombi, which completely

obliterate the capillaries (Fig. 1b, enlarged insert). Con-

cerning the late exudative stage, the epithelial lining is thin

and covered by laminated paucicellular HM. The alveolar

septa are enlarged and contain numerous inflammatory

cells.

After approximately 7 days, the organizing or prolifera-

tive stage is observed with increased interstitial cellularity.

The number of large cuboidal cells, which resemble epi-

thelial type II cells and might represent a stem cell population

of the lung (Fig. 1c and d), increases strikingly. The com-

position of the interstitial cells changes; neutrophils are

partly replaced by macrophages, lymphocytes, and plasma

cells. The interstitium is organized by the proliferation of

connective tissue, persisting edema, and convolutes of loose

fibrous tissue without capillaries. There is a strong diminu-

tion of the microvasculature that is sometimes compressed

by the surrounding tissue. Myofibroblasts that express

vimentin and a-smooth muscle actin (a-SMA) are progres-

sively observed in interstitium and then in airspaces, with a

maximum in the early proliferative stage [63]. The late

proliferative phase shows easily identifiable proliferating

intra-septal and/or intra-alveolar myofibroblasts (airspace

fibroplasia) and production of new matrix substances with

the doubling of lung collagen in 2 weeks.

After a few more days, the fibrotic phase shows wide

connective tissue area interspread between alveolar septa.

Bulk tissue masses formed by folded up septa and col-

lapsed alveoli surround unusually wide airspaces, which

originated mostly from widened alveolar ducts or respira-

tory bronchioles. Some airspace is enlarged due to tissue

destruction. The histology is characterized by enlarged

fibrotic septa and laminated intra-alveolar fibrosis.

ROS are increasingly considered key substances in the

initiation of endothelial damage characteristic of ARDS

and are responsible for most of the clinical symptoms of

this syndrome. There are several causes that increase oxi-

dative stress in ARDS such as breathing high inspiratory

oxygen concentration. However, the large majority of

oxidants are generated by phagocytic cells transmigrating

into the lungs. Neutrophils are crucial since they appear

early in histological specimens and are strongly increased

in the BAL. They release many inflammatory mediators

that include chemokines, cytokines, and proteases [61].

EII

HM

E

HMTN

En

A B

C D

MF

EII

IN

EII

TN

En

Fig. 1 Histological hallmarks of acute respiratory distress syndrome

during the exudative and the proliferative phases. a, b Lung sections

stained with hematoxylin and eosin (H&E) obtained from biopsy of

ARDS subjects during the exudative phase. a Deposition of hyaline

membranes (HM) on the epithelial side of the basement membrane.

At this stage, the presence of detached epithelial type II cells from the

alveolar wall (EII) is also apparent. b The presence of the interstitial

edema (E). The necrosis of endothelial cells (En) and the formation of

thrombus associated with the margination of neutrophil (TN) are also

obvious at this stage. Original magnification 9400 (a and b), 9500

for enlarged insert. c, d Lung sections stained with (H&E) obtained

from biopsy of ARDS subjects during the proliferative phase. c, d The

evident hyperplasia of epithelial type II cells (EII) and an extended

zone of interstitial (IN) fibro-proliferation. Note the presence of

myofibroblasts in the parenchyma (MF). Original magnifications

9200 (c), 9400 (d)


123

The activation of the neutrophils may occur at remote sites

and/or by circulating cytokines, resulting in ROS release

and increased pulmonary vascular permeability. However,

neutrophils are not mandatory for the development of

ARDS, because it can occur in neutropenic patients. Other

key initiators of the pulmonary inflammation in ARDS are

the circulating inflammatory mediators (e.g., TNF-a, IL-

1b, IL-6, IL-8, leukotrienes), as well as changes in the

coagulation system. Alveolar edema resulting from endo-

thelial dysfunction and loss of epithelial integrity reduces

the barrier function. The edema is even increased by the

loss of type II pneumocytes that normally promote fluid

transport out of the alveolus through their apical sodium

pumps. Early loss of surfactant is explained by the damage

of type II epithelial cells, which produce surfactant and by

its neutralization by protein-rich edema fluid. This con-

tributes to alveolar collapse, intrapulmonary shunt, and

hypoxemia. The hypoxic pulmonary vasoconstriction is

impaired by endothelial and smooth muscle cell dysfunc-

tion. This may, with the association of microthrombi,

contribute to the development of secondary pulmonary

hypertension.

Role of NOX enzymes in ALI/ARDS

Whereas the mechanisms that initiate ALI/ARDS remain

unclear, there is some evidence that ROS generated by

NOX enzymes participate in the pathogenesis of this syn-

drome. The study of NOX enzyme contribution in the

patho-mechanisms of ALI/ARDS in human is difficult due

to the heterogeneity of causes and the paucity of biological

samples (biopsies, autopsies, BALF). Animal models

reproduce major clinical features of ALI/ARDS observed

in humans including the loss of the alveolar-capillary

barrier with the damage of both epithelial and endothelial

cells and the inflammatory cell influx. In this way, all these

models provide key elements to study the role of the NOX

family during ALI/ARDS. In the next part, we will

examine their involvement in different mouse models of

ALI/ARDS that mimic neutrophilic infiltration and lung

injury in sepsis-like models and the damage of the alveolar-

capillary barrier.

Lung inflammation and injury

Histological analysis of lung sections from ARDS patients

as well as BAL obtained in the acute phase of the disease

show massive accumulation of neutrophils [64] and most

acute lung injuries induced in animal models are neutro-

phil-dependent [65–67]. These inflammatory cells produce

high levels of ROS, which are thought to increase the

inflammatory processes and tissue injury in septic shock

syndrome [68–70]. In addition, ROS participates in the

modulation of cell-signaling pathways that activate tran-

scription factor of redox-sensitive pro-inflammatory

mediators such as NF-jB [71, 72]. The studies concerning

the effect of ROS generated by NOX enzymes in acute

inflammatory responses and lung injury following Esche-

richia coli and lipopolysaccharide (LPS) challenges in

mice are controversial [37, 71, 73, 74]. Indeed, some

studies have shown that the absence of p47phox (a regula-

tory subunit essential for NOX2 activation) did not

contribute to LPS-induced lung damage, vascular leakage,

and infiltration of neutrophils and monocytes in mice [75].

Swain et al. [76] did not observe any improvement of

pulmonary lung injury in gp91phox-deficient mice during

pneumocystis pneumonia. By contrast, it has been dem-

onstrated that LPS-induced inflammation and lung injury

was inhibited but also in some cases increased in NOX-

deficient mouse models. The absence of p47phox and

gp91phox has been associated with enhanced inflammatory

gene expression, lung neutrophil recruitment, and mouse

survival after LPS challenge [77, 78]. On the other hand,

LPS-induced lung inflammation was reduced in mice

deficient for Nrf2, a regulator of antioxidant defenses, in

absence of p47phox or gp91phox [37]. Moreover, ROS pro-

duction restricted to macrophages from Nrf2-deficient mice

was blunted by the absence of gp91phox after LPS challenge

[37]. Similarly, inflammatory response induced by live

Escherichia coli or LPS was reduced in lung tissues of

p47phox- and gp91phox-deficient mice [71, 73]. Sadikot et al.

[74] has reported that NF-jB activation and TNF-a levels

were decreased in p47phox-deficient mice after Pseudomo-

nas aeruginosa infection and a recent study showed that

ROS generated by NOX2 in neutrophils were involved in

TNF-a-induced acute lung injury [38] and participated in

inflammatory response through the activation of NF-jB

[36]. These results support the notion that ROS generated

by NOX2 play a critical role in the induction of inflam-

matory responses and tissue injury in sepsis.

The family of Toll-like receptors (TLR), which to date

contains ten members, recognizes specific molecules con-

served among microorganisms and pathogens, and plays an

important role in initiating the inflammatory response [79].

Emerging evidence demonstrates that NOX enzymes

modulate Toll-like receptor 4 (TLR4) and TLR2 signaling

not only in neutrophils and macrophages but also in other

cells. Lipopolysaccharide specifically binds to LPS-binding

protein (LBP) and forms a complex that activates the TLR4

receptor of macrophages and others cells. This interaction

triggers the activation of Ijb kinase and the mitogen-acti-

vated protein kinase kinases (MAPKK), which in turn

activate NF-jB and AP1, respectively [80]. Activated

NF-jB and AP-1 translocate into the nucleus where they

bind to DNA promoter regions and induce the transcription


123

of inflammatory genes. It has been shown that in neutrophils,

NOX2-derived ROS regulate the TLR4-mediated activa-

tion of NF-jB. In addition, in endothelial cells, NOX2 also

contributes to TLR2 gene activation in response to LPS

[36, 81]. A recent study demonstrated that ROS generated

by NOX2 in neutrophils mediates high mobility group box

1 (HMGB1)/TLR4 signaling and tissue damage after

hemorrhagic shock/resuscitation in mice [82]. Besides

NOX2, NOX4 is able to directly interact with TLR4 and

mediate ROS generation and NF-jB activation in

HEK293T cells [83]. More recently, NOX4 has been

shown to mediate LPS-induced NK-jB-dependent IL8,

MCP-1 and ICAM-1 gene expression in human aortic

endothelial cells [84] and interferon regulatory factor

(IRF)-3 transcription factor activation in U373/CD14 cells

[85]. NADPH oxidase4 is also able to activate AP-1 and

subsequent CXCR6 expression, after LPS challenge in

human aortic smooth muscle cells [86]. Similar to NOX4,

the presence of crosstalk between NOX1 and TLR4 was

suggested by the observation that LPS derived from Heli-

cobacter pylori increases ROS production and NOX1

expression in guinea pig gastric pit cells through a TLR4

signaling pathway [87]. All these results suggest that NOX

family plays an important role in the activation of TLR4

signaling pathways (including NF-jB, IRF-3 and AP1) in

response to LPS. Nevertheless, the molecular mechanism

linking TLR4 to NOX1 remains unclear and the function of

NOX1 and NOX4 in TLR-mediated signaling in vivo need

to be elucidated using either knock-out mice or RNA

interference strategy.

Endothelial and epithelial targets

Alveolar cell death has been reported extensively in

humans and in experimental models of acute lung injury

[88]. Indeed, in the acute phase, the presence of edema in

the air spaces and hyaline membrane deposits are direct

consequences of alveolar-capillary barrier damage. Epi-

thelial type II cells after being initially injured, often by an

unknown stimulus, proliferate in order to repair the dam-

aged epithelium [89]. To date, it is not known whether the

alveolar-capillary barrier integrity depends preferentially

on the endothelial or the epithelial side in acute lung

damage and which are the signaling pathways involved in

alveolar cell death. Nevertheless, there is evidence that

both epithelial and endothelial cells are damaged by ROS-

dependent mechanisms and in particular by NOX-depen-

dent ROS generation.

Endothelial cell target

The injury of endothelial cells is mostly studied in sepsis-

like models using systemic injection of LPS or TNF-a.

During ALI, the endothelium undergoes large transforma-

tions in terms of expression of adhesion molecules, tight

junctions, and ROS-producing enzymes [90]. In this con-

text, NOX-derived ROS participates in the damage of

endothelial cells either by the direct activation of endo-

thelial signaling or via neutrophils or macrophages.

Lipopolysaccharide can directly increase ROS genera-

tion through the modulation of NOX enzymes in

endothelial cells [91]. A recent study demonstrates that in

these cells, stimulation by LPS leads to the activation of

IL8, which in turn regulates the expression and the activity

of NOX1 and contributes to the progression of the sepsis

cascade. These data suggest that LPS/IL8 signaling is

NOX1-dependent in endothelial cells [92]. In addition to

NOX1, NOX4, which is also expressed in endothelial cells,

is responsible for LPS/TLR4-induced ROS generation and

gene expression of chemokines such as IL8, MCP-1, and

intracellular adhesion molecule-1 (ICAM-1) in human

aortic endothelial cells [84]. The authors also demonstrated

that the specific inhibition of NOX4, by siRNA strategy,

contributes to the decrease of LPS-induced migration and

adhesion of monocytes to endothelial cells [84].

Thus, besides the direct effect of LPS on NOX activa-

tion in endothelial cells, excessive production of ROS by

NOX enzymes located in inflammatory cells has been

associated to endothelial cell damage in sepsis. Fan et al.

[34, 81] and others demonstrated that in endothelial cells,

LPS/TLR4-induced NF-jB and TLR2 gene activation is

dependent on NOX2 located in neutrophils. In addition,

neutrophilic NOX2 contributes to TNF-a-induced NF-jB-

dependent lung inflammation and endothelial cell injury in

mice [38] and participates in the activation of NF-jB and

the induction of TLR2 in endothelial cells [36]. More

recently, Farley et al. [41] reported that co-culture of

p47phox and gp91phox-deficient macrophages with pul-

monary microvascular endothelial cells stimulated with a

mix of cytokines such as TNF-a, IL1-b, and IFN-c led to a

significant decrease in endothelial cell injury, supporting

the concept that ROS produced by phagocytic NOX2 play

a crucial role in the injury of endothelial cells.

Epithelial cell target

Although the epithelial barrier after being injured by an

unknown stimulus is mostly able to repair, the persistence

of a lung injury leads to the development of fibrosis. It is

considered that epithelial cell death is crucial not only in

the weakening of the alveolar-capillary barrier, but also in

lung abnormal repair, which leads to pulmonary fibrosis

[93]. While the pathogenesis of pulmonary fibrosis, a lethal

lung disorder characterized by abnormal lung repair, is

unknown, it involves early inflammatory steps and late

fibrotic changes with proliferation of fibroblasts and their


123

differentiation into myofibroblasts [94, 95]. Intratracheal

instillation of bleomycin in mice, a well-known charac-

terized model to study initial lung epithelial injury and

subsequent fibrosis, mimics ALI/ARDS features occurring

during the late proliferative and fibrotic phase. In this

model, NOX-dependent ROS are not only responsible for

initial epithelial damage but also for the differentiation of

fibroblasts into myofibroblasts, the hallmark of the disease.

Several studies have shown that TGF-b1 increases ROS

levels by up-regulating NOX4 expression in lung fibro-

blasts and induces their differentiation into myofibroblasts

[8, 96, 97]. Interestingly, in human idiopathic pulmonary

fibrosis (IPF), NOX4 has been detected in myofibroblasts

of late fibrotic scars, suggesting a possible role of NOX4 in

the development of organized fibrosis, and was also

detected in the alveolar proliferative epithelium of IPF

lungs adjacent to fibroblastic foci. In mice, we demon-

strated that NOX4 deficiency as well as acute treatment

with NOX inhibitors blunted TGF-b1-induced alveolar

epithelial cell death and prevented subsequent pulmonary

fibrosis [7].

The role of NOX2 and NOX1 in bleomycin-induced

lung fibrosis has also been investigated in NOX2 and

NOX1-deficient mice. Only a moderate protection from

bleomycin-induced lung fibrosis was observed in NOX2-

deficient mice [98]; however, extrapolation to human IPF is

difficult as inflammation might not be as prominent in

humans compared to mice. We found that NOX1-deficient

mice were not protected from bleomycin-induced fibrosis

(personal data). Finally, NOX4 rather than NOX1 and

NOX2 could be a good candidate for the treatment of

ARDS/ALI patients during both the acute and the prolif-

erative stage.

Epithelium and endothelium targets

Aspiration of the gastric content is considered to be an

important cause of ALI/ARDS. In addition to the low pH,

the gastric content contains particulate bacterial material,

which contributes to lung injury [99]. The intratracheal

instillation of hypochlorite (HCl) is a well-used model for

inducing lung injury secondary to gastric acid aspiration in

mice. Aspiration-induced lung injury, which depends on

neutrophilic influx into the alveolar space, is characterized

by the damage of both epithelial and endothelial cells

leading to alveolar hemorrhage and edema. Some studies

have implicated ROS as a key element in the pathogenesis

of ALI/ARDS following gastric content aspiration in

mouse models [100], but to date, only one has demon-

strated the role of NOX enzyme in this context. Indeed,

exposure of p47phox-deficient mice to HCl leads to

increased pulmonary neutrophilic infiltration, alveolar-

capillary barrier leakage, and enhanced level of pro-

inflammatory cytokine compared to WT mice [101], sug-

gesting a protective role of NOX2 in HCl-induced lung

injury by modulating the inflammatory response.

Mechanical ventilation (MV) is the unique strategy used

in patients with acute hypoxemic respiratory failure to

improve arterial oxygenation and their survival [102].

However, this therapy provokes tissue injury due to

mechanical stretch (MS). Mechanical ventilation associ-

ated with alveolar barrier overstretching contributes to

neutrophilic infiltration, release of pro-inflammatory cyto-

kines, and lung injury [103]. The cellular mechanisms

involved in MV-induced lung injury (MVILI) and

-inflammation remain unknown. A high level of ROS is

thought to be one potential initiating signal in response to

MV following mechanical stress. Indeed, treatment with

N-acetyl cysteine (NAC) attenuates MV-induced neuro-

philic influx into alveolar spaces and reduces epithelial cell

apoptosis in rats [104, 105]. Besides mitochondrial

enzymes, NOXes have been shown to contribute to ROS

production in response to mechanical stress in different

cells such as endothelial cells, epithelial cells, and vascular

smooth muscle cells [106–111]. It has been described that

NOX activation was associated with a membrane translo-

cation of p47phox in smooth muscle cells (SMC) [108, 109].

On the other hand, exposure of vascular SMCs to MV

leads to p47phox membrane translocation followed by an

increased NOX1 mRNA expression and ROS production

[108], suggesting a role for NOX1 in MVILI. Some studies

also demonstrated that ROS produced by NOX enzymes

participated in cyclic stretch-induced vascular remodeling

in SMC via matrix metalloproteinase-2 activation [108].

The NOX isoform involved in MV-induced lung injury and

the NOX-dependent signal transduction pathways need to

be clarified.

Hyperoxia-induced acute lung injury is one of the most

relevant models of oxidative stress and alveolar cell death,

which is not closely linked to the magnitude of the

inflammatory response. In rodents and in alveolar cell

culture, oxygen toxicity (100 % O2) has been used as a

well-established model of lesions mimicking the acute

phase of ALI/ARDS and for studying direct alveolar

damage induced by high levels of oxidants. It was first

explored in rats and later extensively characterized in mice

[112–114]. During the initiation phase (usually lasting for

48 h), only subtle changes can be detected, such as the

arrest of cell replication, and lesions are not evident on

light microscopy. This phase is followed by diffuse alve-

olar damage with hyaline membrane deposition and

extensive death of alveolar cells (mainly endothelial and

epithelial cells) associated with a generally mild inflam-

matory response, which can vary according to the species

[115]. Alveolar cell death has been shown to be directly

related to increased generation of oxidant in hyperoxic


123

condition [116]. Reactive oxygen species can be generated

by mitochondrial chain transport as well as by NADPH

oxidase enzymes [117]. In vitro studies have shown that the

diphenyleneiodonium (DPI), a non-specific inhibitor of

NOX isoforms, was effective in reducing hyperoxia-

induced ROS generation in a pulmonary epithelial cell line

(MLE-12) and in primary pulmonary type II cells [117–

119]. Recently, our laboratory demonstrated that NOX1,

which is highly expressed in lungs, plays a crucial role in

hyperoxia-induced acute lung injury [17]. NADPH oxi-

dase1-deficient mice exposed to hyperoxia exhibited

reduced pulmonary edema, hyaline membrane deposition,

and alveolar-capillary damage. Indeed, in situ lung cell

death was markedly decreased in NOX1-deficient mice and

paralleled with decreased ROS production and cell death in

endothelial and epithelial cells. The phosphorylation of

both c-Jun N-terminal kinase (JNK) and extracellular sig-

nal-regulated kinase (ERK), as well as caspase-3 activation

were decreased in lung homogenates. All these results

demonstrate a role for NOX1 in hyperoxia-mediated bar-

rier dysfunction; however, the question of the contribution

of NOX1 restricted to the epithelial side or to endothelial

side or both in ALI/ARDS and its precise cellular signaling

pathway is still open.

As stated above, the integrity of the alveolar-capillary

barrier depends not only on the epithelium but also on the

endothelium. Recent studies have shown increased NOX1

mRNA expression in mouse lung endothelial cells after

hyperoxic condition [17, 28] as well as NOX1 contribution

to endothelial cell death [17]. The involvement of NOX4 in

hyperoxic cultured endothelial cells (EC) has also been

investigated. NADPH oxidase4 mRNA expression is

increased in hyperoxia [28] through direct modulation of

gene transcription. Indeed, direct interaction between nrf2

transcription factor and NOX4 promoter has been reported.

The authors demonstrated that hyperoxia increased the

recruitment- and the binding of nfr2 to endogenous NOX4

promoter via antioxidant response element (ARE) in pul-

monary endothelial cells [44]. Hyperoxia also regulates

NOX activation in part by ERK1/2 and p38 MAPK [117],

but also by an Src-dependent tyrosine phosphorylation of

p47phox [29]. It has also been proposed that NOX2 could be

activated by tyrosine phosphorylation of cortactin and

p47phox translocation following hyperoxia in ECs derived

from human pulmonary artery [120]. Recently, Pendyala

et al. [28] demonstrated the contribution of NOX4 in

endothelial dysfunction. They showed that the transfection

of a NOX4-specific siRNA in HPAEC attenuated hyper-

oxia-induced migration and capillary tube formation.

Therefore, we have now convincing evidence of NOXs

activation (NOX1, 2 and 4) by oxidative stress in murine

and human endothelial cells [17, 29, 117, 120, 121].

Although we have less evidence, we could also hypothesize

that NOX4 participates in epithelial cell damage in ALI/

ARDS. Indeed, we recently reported that NOX4 mRNA

was expressed in primary type II epithelial cells and

mediated TGF-b1-induced epithelial cell death [7]. These

results suggest that NOX4 contributes not only to the

dysfunction or death of endothelial cells, but it might be

involved in the alveolar-capillary disruption observed in

ALI/ARDS. However, all these experiments were per-

formed essentially in cell cultures and should be confirmed

first by using NOX4-deficient mice or/and transfection of

NOX4-specific siRNA and then in humans.

Controversial studies concerning the role of NOX2 in

hyperoxia-induced lung injury have been reported.

Pendyala showed that NOX2-deficient mice exposed to

acute hyperoxia developed attenuated pulmonary edema,

lung fibrosis, and weak inflammatory response [122]. By

contrast, we found that NOX2-deficient mice exposed to

hyperoxia were not protected and display a huge neutrophil

influx in BAL, alveolar cell death, and lung injury [17],

suggesting that NOX2 does not mediate alveolar-capillary

disruption in hyperoxia. Indeed, neutrophil or macrophage

depletion did not change lung damage in hyperoxic lung

injury [123, 124]. Similarly, NOX2-deficient mice exposed

to 48 h of hyperoxia following acid aspiration showed a

greater amount of neutrophils compared to WT mice,

without modification of lung injury [73].

NOX as treatment of ALI/ARDS

As largely described above, NOX inhibitors might have

potential in vivo use in ARDS. However, the multiplicity

of lung cells combined with the cellular and functional

specificity of the different NOX isoforms makes this

approach delicate. Moreover, the measurement of NOX

activity is often indirect since it is evaluated by the dosage

of ROS-derived products using colorimetric or fluorescent

probes. One must be aware that measuring ROS or derived

products levels might not only be due to ROS production

by NOX enzymes but also by other ROS-producing

enzymes. Therefore the specific efficacy of NOX inhibition

can be difficult to prove.

Today, therapeutic intervention for ALI/ARDS consists

of protecting the lung by using adaptive mechanical ven-

tilation and oxygenation and thus limiting mortality [125].

Lung-protective mechanical ventilation with lower tidal

volumes in patients not suffering from acute lung injury: a

review of clinical studies. This strategy was elaborated

according to the results obtained in clinical trials and in

experimental animal models [126]. Some studies have

suggested that subgroups of patients may benefit from

targeted therapeutic interventions. Most promising is the

differentiation between patients in early versus late-phase


123

ARDS, direct versus indirect lung injury, and patients with

altered coagulation. A high dose of corticosteroid admin-

istration did not improve mortality, whereas low to

moderate doses appear to be harmful if initiated later and

are of unclear benefit [127, 128]. Surfactant supplementa-

tion was shown to be helpful only in pediatric patients with

direct lung injury [129] and anticoagulants may be suc-

cessful in the subgroup of patients with vascular disease

[130]. There is an interest in developing NOX inhibitors,

since they can act for some of them in the early phase and

for other in the fibro-proliferative phase. Table 2 summa-

rizes the expected effects of the potential NOX inhibitors

for the treatment of ARDS.

NADPH oxidase1 inhibition might be useful in the acute

phase of ALI/ARDS since it interferes with endothelial and

epithelial cell death either by decreasing oxidative stress-

induced genotoxicity or by affecting MAPK signaling

pathways [17]. However, this study was performed only in

mice and evidence in humans is still required. The possi-

bility that NOX1 inhibition can affect TNF activation

might also be interesting in case of ARDS due to sepsis

[22].

NADPH oxidase2 inhibition effects are more complex

and the results are somehow controversial. NADPH oxi-

dase2 is present mainly in phagocytic cells, but also in a

great amount in endothelial cells. Indeed, changing

phagocyte killing might be dangerous in situations of

ARDS due to sepsis or to unknown origin even if ROS-

produced by NOX2 in other cells might prevent concom-

itantly. In this case, a tagged-cell inhibitor would be ideal,

but at the present time rather difficult.

More promising would be the inhibition of NOX4,

which is present in several lung cells epithelial and fibro-

blasts. Several studies have shown a very robust effect in

decreasing epithelial cell death initiated by TGF-b1 and

myofibroblast differentiation [7, 8]. Moreover, NOX4

action is upstream of the pleiomorph effects of TGF-b1,

and therefore could be more efficient in blocking the TGF-

b1 deleterious cascade. We can also hypothesize, as NOX4

is strongly expressed in epithelial cells and its signaling

potentiates cell death induced by TGF-b1, that NOX4-

specific inhibition could prevent the alveolar-capillary

disruption in the ARDS early phase [7]. NADPH oxidase4

is also involved in TLR4 signaling mediated by LPS and

might therefore participate in endothelial dysfunction [84].

This study has been performed in vitro, and more in vivo

data are needed before envisaging therapeutic possibilities.

Conclusions

NADPH oxidase enzymes, which are widely expressed in

different lung cell types, participate not only in the main-

tenance of physiological processes in lungs but also

contribute to the pathogenesis of acute lung diseases such

as ALI and ARDS. The multiplicity of the well-charac-

terized animal models mimicking ARDS/ALI, the use of

NOX-deficient mice and in vivo siRNA transfection strat-

egies allowed to explore NOX-dependent cellular and

molecular mechanisms involved in the development of the

disease and finally envisage new therapeutic approaches.

Developing NOX inhibitors could therefore be a promising

treatment concept for ARDS/ALI. Nevertheless, at the

present time, no direct method for measuring specifically

NOX-dependent ROS generation has been developed to

prove the efficacy of NOX inhibitors and specific inhibitors

for one single NOX isoform are not available. Whether in

some case it might be useful to target two different iso-

forms concomitantly, such as in early phase, in other

situations such as ARDS induced by sepsis, this could be

deleterious due to combined unwarranted secondary

effects. Thus, further in vivo studies concerning NOX

inhibitors are necessary to prove their clinical utility in the

management of ALI/ARDS.

Table 2 Summary of potential effect of NOX inhibitors in ALI/ARDS

NOX isoform

inhibitors

ARDS/ALI clinical stages Target cells Expected effects Secondary effects

NOX1 Acute stage: alveolar-

capillary barrier disruption

Epithelial and

endothelial cells

Decreased cell death (genotoxicity, MAPK signaling,

TNF-RI-JNK signaling)

N.D

NOX2 Acute stage: inflammation/

endothelial cell injury

Macrophages/

neutrophils

Decreased inflammatory response, endothelial cell

death, crosstalk with TLR4 signaling

Increased

susceptibility to

infection

Endothelial cells Decreased cell death, crosstalk with TLR2 signaling N.D

NOX4 Acute stage: alveolar-

capillary barrier disruption

Epithelial cells Decreased cell death (genotoxicity) interference with

TGF-b signaling

N.D

Acute stage: inflammation/

endothelial cell injury

Endothelial cells Decreased cell death,TLR4 crosstalk signaling N.D

Fibro-proliferative stage Myofibroblasts Decreased proliferation and differentiation,

interference with TGF-b1 signaling

N.D


123

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NOX enzymes: potential target for the treatment of acute lung injury